A method for determining kinetic and thermodynamic parameters at solid-liquid interfaces

Abstract

The present invention discloses a method for determining kinetic and thermodynamic parameters on a solid-liquid interface. According to the frequency-time curves corresponding to a plurality of ligand solutions with different concentrations to be tested and the frequency-time curves of another ligand solution with different concentrations to be tested at two different temperatures, the kinetic and thermodynamic parameters (binding / dissociation equilibrium constant K of a nucleic acid aptamer and its ligand on a solid-liquid interface) of the nucleic acid aptamer and its ligand are obtained by analyzing and calculating the frequency-time curves output by a microcantilever sensor. A / K D , Gibbs free energy △G°, surface coverage θ, association / dissociation rate constant k a / k d , the reaction activation energy E a ). This method overcomes the shortcomings of traditional methods such as expensive instruments, cumbersome operations, and single parameter determination. It has the advantages of high sensitivity, low cost, simple and fast operation, no need for labeling and calibration, can effectively avoid systematic errors, and can measure related parameters at one time. It is suitable for the determination of the kinetic and thermodynamic parameters of various nucleic acid aptamers and their ligands.

Description

Technical Field

[0001] The invention relates to the field of molecular dynamics / thermodynamics technology, and in particular to a method for measuring kinetic and thermodynamic parameters of biochemical reactions on a solid-liquid interface. Background Art

[0002] Nucleic acid aptamers are synthetic RNA or DNA receptor molecules, and a variety of target substances (small molecules, proteins and even cells) can become their ligands. Because nucleic acid aptamers are chemically stable, easy to synthesize, and can be modified according to needs, they have high specificity and affinity for their ligands. Nucleic acid aptamers have become a commonly used molecular recognition element in the development and application of biosensors.

[0003] When using biosensors to detect analytes in solutions, the place where reactions, energy transfer, and signal conversion occur is the solid-liquid interface. Therefore, quantitative studies on the kinetics / thermodynamics of biochemical reactions at the solid-liquid interface can study biosensing mechanisms, improve sensor performance, evaluate interface design and material optimization, and guide the design and manufacture of biosensors. Although a large number of studies have been conducted on the kinetics and thermodynamics of nucleic acid aptamers and their ligands in solutions, the parameters obtained from the solution cannot describe the kinetics and thermodynamics of biochemical reactions at the interface due to the energy barriers generated by the diffusion layer and the steric hindrance effect of the capture probe at the solid-liquid interface; however, the existing kinetics and thermodynamics research methods at the solid-liquid interface are often limited by factors such as signal labeling and temperature range. Therefore, the development of biointerface sensing, especially the biosensor system based on the reaction of nucleic acid aptamers and ligands, urgently needs to provide a method for rapidly determining the kinetics and thermodynamic parameters of nucleic acid aptamers and ligands at the solid-liquid interface, so as to systematically study the kinetics and thermodynamics mechanisms and physicochemical processes at the solid-liquid interface.

[0004] In view of this, it is necessary to provide a method for measuring the kinetic and thermodynamic parameters at the solid-liquid interface to solve the above technical problems. Summary of the invention

[0005] In view of the limitations of traditional methods for measuring kinetic and thermodynamic parameters on interfaces, the purpose of the present invention is to provide a more direct, convenient and comprehensive method, which can directly and quickly obtain the required kinetic and thermodynamic parameters based on the frequency-time curve, use a microcantilever sensor as a detection platform, track the reaction occurring at the solid-liquid interface in real time, and extract multiple kinetic / thermodynamic parameters at one time, without the need for modification or labeling of the analyte, and without mass sensitivity calibration, thereby improving measurement efficiency, effectively avoiding measurement errors, and reducing measurement costs.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides a method for measuring kinetic and thermodynamic parameters at a solid-liquid interface, comprising the following steps:

[0008] S100. Provide a frequency-time graph corresponding to each of a plurality of ligand solutions of different concentrations to be tested and a frequency-time graph of another ligand solution of a concentration to be tested at two different temperatures, wherein the plurality of ligand solutions of different concentrations to be tested include at least three ligand solutions of different concentrations to be tested;

[0009] S200. Calculate the kinetic and thermodynamic parameters at the solid-liquid interface according to the frequency-time curves corresponding to the various ligand solutions of different concentrations to be tested, wherein the parameters include at least the binding equilibrium constant K A , dissociation equilibrium constant K D , Gibbs free energy △G°, surface coverage θ, binding rate constant k a and the dissociation rate constant k d ;

[0010] S300. Calculate the reaction activation energy E according to the frequency-time curve of another ligand solution of the concentration to be tested at the two different temperatures. a .

[0011] Furthermore, the kinetic and thermodynamic parameters at the solid-liquid interface are calculated based on the frequency-time curves corresponding to the various ligand solutions of different concentrations to be tested, and the parameters at least include the binding equilibrium constant K A , dissociation equilibrium constant K D , Gibbs free energy △G°, surface coverage θ, binding rate constant k a and the dissociation rate constant k d include:

[0012] S201. Reading frequency change values ​​△f corresponding to the various different concentrations of the ligand to be tested according to the frequency-time curves corresponding to the various different concentrations of the ligand to be tested;

[0013] S202. Calculate the binding equilibrium constant K of the reaction according to the frequency change values ​​△f corresponding to the various concentrations of the ligands to be tested. A ;

[0014] S203. According to the binding equilibrium constant K of the reaction A , calculate the dissociation equilibrium constant K of the reaction D ;

[0015] S204. According to the binding equilibrium constant K of the reaction A , calculate the Gibbs free energy of the reaction △G°;

[0016] S205. According to the binding equilibrium constant K of the reaction A , the surface coverage θ corresponding to various different concentrations of the ligand to be tested is calculated;

[0017] S206. Calculate the binding rate constant k of the reaction according to the frequency-time curves and the corresponding surface coverage θ corresponding to the various ligand solutions of different concentrations to be tested. a ;

[0018] S207. According to the binding rate constant k of the reaction a , the dissociation rate constant k is calculated d .

[0019] Furthermore, the binding equilibrium constant K of the reaction is calculated based on the frequency change values ​​Δf corresponding to the various concentrations of the ligand to be tested. A include:

[0020] According to the frequency change values ​​△f corresponding to the various concentrations of the ligands to be tested, the formula Calculate the binding equilibrium constant K of the reaction A .

[0021] Where [L] is the concentration of the ligand to be tested, and A is a constant reflecting the maximum number of reactive sites on the interface.

[0022] Further, the binding equilibrium constant K according to the reaction A , calculate the dissociation equilibrium constant K of the reaction D include:

[0023] According to the binding equilibrium constant K of the reaction A , through the formula K D =1 / K A Calculate the dissociation equilibrium constant K of the reaction D .

[0024] Further, the binding equilibrium constant K according to the reaction A , the calculated Gibbs free energy of the reaction △G° includes:

[0025] According to the binding equilibrium constant K of the reaction A , through the formula △G°=-RTlnK A Calculate the Gibbs free energy of the reaction, △G°.

[0026] Wherein, R is the ideal gas constant, R = 8.314 J / (mol·K), and T is the absolute temperature (K) at which the experiment is conducted.

[0027] Further, the binding equilibrium constant K according to the reaction A, the surface coverage θ corresponding to various different concentrations of the ligand to be tested is calculated, including:

[0028] According to the binding equilibrium constant K of the reaction A , through the formula The surface coverage θ corresponding to various different concentrations of the ligand to be tested was calculated.

[0029] Furthermore, the binding rate constant k of the reaction is calculated based on the frequency-time curves and the corresponding surface coverage θ corresponding to the various ligand solutions with different concentrations to be tested. a include:

[0030] S2061. According to the frequency-time curves corresponding to the various ligand solutions with different concentrations to be tested, the slopes b of the frequency-time curves corresponding to the various ligand solutions with different concentrations to be tested are read;

[0031] S2062. According to the frequency-time curve slope b corresponding to the plurality of different ligand concentrations to be tested, the frequency change value △f corresponding to the plurality of different ligand concentrations to be tested and the corresponding surface coverage θ, the formula The binding rate constant k of the reaction was calculated a .

[0032] Further, the binding rate constant k according to the reaction a , the dissociation rate constant k is calculated d include:

[0033] According to the binding rate constant k of the reaction a , through the formula k d =k a / K A The dissociation rate constant k was calculated. d .

[0034] Furthermore, the two different temperatures include a first preset temperature T1 and a second preset temperature T2, and the reaction activation energy E is calculated based on the frequency-time curve of another ligand solution of the concentration to be tested at the two different temperatures. a include:

[0035] S301. According to the frequency-time curves of another ligand solution of the measured concentration at the two different temperatures, obtain the slope b1 of the frequency-time curve at the first preset temperature T1 and the slope b2 of the frequency-time curve at the second preset temperature T2;

[0036] S302. Calculate the activation energy E according to the first preset temperature T1, the second preset temperature T2, the slope b1 of the frequency-time curve at the first preset temperature T1, and the slope b2 of the frequency-time curve at the second preset temperature T2. a .

[0037] Further, the activation energy E is calculated based on the first preset temperature T1, the second preset temperature T2, the slope b1 of the frequency-time curve at the first preset temperature T1, and the slope b2 of the frequency-time curve at the second preset temperature T2. a include:

[0038] According to the first preset temperature T1, the second preset temperature T2, the slope b1 of the frequency-time curve at the first preset temperature T1, and the slope b2 of the frequency-time curve at the second preset temperature T2, the formula Calculate the activation energy E a .

[0039] Wherein, R is the ideal gas constant, R = 8.314 J / (mol·K).

[0040] The implementation of the present invention has the following beneficial effects:

[0041] 1. The present invention can directly and quickly obtain the required kinetic and thermodynamic parameters according to the frequency-time curve, and the method for determining the required data curve is simple and fast, without the need for complex instrument operation. Based on the micro-weighing detection mode of the micro-cantilever sensor, direct detection can be performed without labeling the ligand in the solution;

[0042] 2. The method proposed in the present invention can extract multiple kinetic and thermodynamic parameters at one time based on the data collected by a microcantilever sensor, thus avoiding the systematic errors introduced by multiple experiments and multiple instruments;

[0043] 3. The method proposed in the present invention does not require mass sensitivity calibration of the microcantilever sensor, which simplifies the experimental steps and improves the measurement efficiency;

[0044] 4. The microcantilever sensor used in the present invention has extremely high sensitivity, overcoming the limitation that other methods can only detect macromolecules, and is used to study the interaction mechanism between small molecules with low molecular weight and their nucleic acid aptamers at the interface. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the technical solutions and advantages in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0046] Figure 1 It is a flow chart of a method for determining kinetic and thermodynamic parameters at a solid-liquid interface provided by an embodiment of the present invention.

[0047] Figure 2 It is a flow chart of step S200 provided in an embodiment of the present invention.

[0048] Figure 3 It is a flow chart of step S300 provided in an embodiment of the present invention.

[0049] Figure 4 It is a schematic diagram of a micro-cantilever sensor detection system provided in an embodiment of the present invention.

[0050] Figure 5 This is the reaction of adenosine triphosphate (ATP) and its nucleic acid aptamer at the solid-liquid interface provided by an embodiment of the present invention, and the frequency-time curve obtained at different ATP concentrations [L] at the same temperature.

[0051] Figure 6 The embodiment of the present invention provides the use of Figure 5 The data obtained were used to calculate the association / dissociation equilibrium constant K A / K D , [L] / △f-[L] curve of Gibbs free energy △G°.

[0052] Figure 7 The embodiment of the present invention provides the use of Figure 5 The data obtained were used to calculate the surface coverage θ, the binding rate constant k a and the dissociation rate constant k d b-[L]·△f / θ curve.

[0053] Figure 8 It is a frequency-time curve of measuring the same ATP concentration at different temperatures provided by an embodiment of the present invention.

[0054] Among them, the corresponding reference numerals are: 1-buffer solution, 2-ligand solution, 3-injection valve, 4-reaction chamber, 41-injection port, 42-outlet, 5-detection chamber, 6-microcantilever chip, 7-temperature control device, 8-constant pressure pump, 9-waste liquid outlet, 10-computer, 11-data acquisition device. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention.

[0056] The present invention provides a method for determining the kinetic and thermodynamic parameters at the solid-liquid interface. Figures 1 to 8 The method for determining the kinetic and thermodynamic parameters at the solid-liquid interface comprises the following steps:

[0057] S100. Provide a frequency-time graph corresponding to each of a plurality of ligand solutions of different concentrations to be tested and a frequency-time graph of another ligand solution of a concentration to be tested at two different temperatures, wherein the plurality of ligand solutions of different concentrations to be tested include at least three ligand solutions of different concentrations to be tested;

[0058] S200. Calculate the kinetic and thermodynamic parameters at the solid-liquid interface according to the frequency-time curves corresponding to the various ligand solutions of different concentrations to be tested, wherein the parameters include at least the binding equilibrium constant K A , dissociation equilibrium constant K D , Gibbs free energy △G°, surface coverage θ, binding rate constant k a and the dissociation rate constant k d ;

[0059] S300. Calculate the reaction activation energy E according to the frequency-time curve of another ligand solution of the concentration to be tested at the two different temperatures. a .

[0060] Furthermore, the kinetic and thermodynamic parameters at the solid-liquid interface are calculated based on the frequency-time curves corresponding to the various ligand solutions of different concentrations to be tested, and the parameters at least include the binding equilibrium constant K A , dissociation equilibrium constant K D , Gibbs free energy △G°, surface coverage θ, binding rate constant k a and the dissociation rate constant k d include:

[0061] S201. Reading frequency change values ​​△f corresponding to the various different concentrations of the ligand to be tested according to the frequency-time curves corresponding to the various different concentrations of the ligand to be tested;

[0062] S202. Calculate the binding equilibrium constant K of the reaction according to the frequency change values ​​△f corresponding to the various concentrations of the ligand to be tested. A ;

[0063] S203. According to the binding equilibrium constant K of the reaction A , calculate the dissociation equilibrium constant K of the reaction D ;

[0064] S204. According to the binding equilibrium constant K of the reaction A , calculate the Gibbs free energy of the reaction △G°;

[0065] S205. According to the binding equilibrium constant K of the reaction A , the surface coverage θ corresponding to various different concentrations of the ligand to be tested is calculated;

[0066] S206. Calculate the binding rate constant k of the reaction according to the frequency-time curves and the corresponding surface coverage θ corresponding to the various ligand solutions of different concentrations to be tested. a ;

[0067] S207. According to the binding rate constant k of the reaction a , the dissociation rate constant k is calculated d .

[0068] like Figure 2 As shown, the binding equilibrium constant K of the reaction is calculated based on the frequency change values ​​△f corresponding to the various concentrations of the ligand to be tested. A include:

[0069] According to the frequency change values ​​△f corresponding to the various concentrations of the ligands to be tested, the formula Calculate the binding equilibrium constant K of the reaction A Where [L] is the concentration of the ligand to be tested, and A is a constant reflecting the maximum number of reactive sites on the interface.

[0070] The binding equilibrium constant K according to the reaction A , calculate the dissociation equilibrium constant K of the reaction D include:

[0071] According to the binding equilibrium constant K of the reaction A , through the formula K D =1 / K A Calculate the dissociation equilibrium constant K of the reaction D .

[0072] The binding equilibrium constant K according to the reaction A , the calculated Gibbs free energy of the reaction △G° includes:

[0073] According to the binding equilibrium constant K of the reaction A , through the formula △G°=-RTlnKA Calculate the Gibbs free energy of the reaction, △G°.

[0074] Wherein, R is the ideal gas constant, R = 8.314 J / (mol·K), and T is the absolute temperature (K) at which the experiment is conducted.

[0075] The binding equilibrium constant K according to the reaction A , the surface coverage θ corresponding to various different concentrations of the ligand to be tested is calculated, including:

[0076] According to the binding equilibrium constant K of the reaction A , through the formula The surface coverage θ corresponding to various different concentrations of the ligand to be tested was calculated.

[0077] The binding rate constant k of the reaction is calculated based on the frequency-time curves corresponding to the various ligand solutions with different concentrations to be tested. a include:

[0078] S2061. According to the frequency-time curves corresponding to the various ligand solutions with different concentrations to be tested, the slopes b of the frequency-time curves corresponding to the various ligand solutions with different concentrations to be tested are read;

[0079] S2062. According to the frequency-time curve slope b corresponding to the plurality of different ligand concentrations to be tested, the frequency change value △f corresponding to the plurality of different ligand concentrations to be tested and the corresponding surface coverage θ, the formula The binding rate constant k of the reaction was calculated a .

[0080] The binding rate constant k of the reaction is a , the dissociation rate constant k is calculated d include:

[0081] According to the binding rate constant k of the reaction a , through the formula k d =k a / K A The dissociation rate constant k was calculated. d .

[0082] like Figure 3 As shown, the two different temperatures include a first preset temperature T1 and a second preset temperature T2.

[0083] The reaction activation energy E is calculated based on the frequency-time curve of the ligand solution of another concentration to be tested at the two different temperatures. a include:

[0084] S301. According to the frequency-time curves of another ligand solution of the measured concentration at the two different temperatures, obtain the slope b1 of the frequency-time curve at the first preset temperature T1 and the slope b2 of the frequency-time curve at the second preset temperature T2;

[0085] S302. Calculate the activation energy E according to the first preset temperature T1, the second preset temperature T2, the slope b1 of the frequency-time curve at the first preset temperature T1, and the slope b2 of the frequency-time curve at the second preset temperature T2. a .

[0086] Further, the activation energy E is calculated based on the first preset temperature T1, the second preset temperature T2, the slope b1 of the frequency-time curve at the first preset temperature T1, and the slope b2 of the frequency-time curve at the second preset temperature T2. a include:

[0087] According to the first preset temperature T1, the second preset temperature T2, the slope b1 of the frequency-time curve at the first preset temperature T1, and the slope b2 of the frequency-time curve at the second preset temperature T2, the formula Calculate the activation energy E a . Wherein, R is the ideal gas constant, R = 8.314 J / (mol·K).

[0088] like Figures 4 to 8 As shown, the method for determining the kinetic and thermodynamic parameters at the solid-liquid interface proposed by the present invention is specifically as follows:

[0089] 1) The amino-modified nucleic acid aptamer (sequence: 5'-ACCTGGGGGAGTATTGCGGAGGAAGGTTTT-NH2-3') was bound to polydopamine microspheres.

[0090] 2) Upload to the detection area of ​​the microcantilever chip through a micro-printer.

[0091] 3) Place the micro-cantilever chip into the detection system and seal it. The detection chip based on micro-cantilever technology has low cost, small size and good repeatability. The micro-weighing detection mode of the micro-cantilever sensor can directly detect the ligand in the solution without labeling it, and output a continuous signal in real time to achieve complete tracking of the reaction process.

[0092] 4) If Figure 4As shown, the injection channel of the injection valve 3 is transferred into the buffer solution 1, the constant pressure pump 8 is turned on, the system becomes negative pressure, the buffer solution passes through the injection port 41, fills the detection cavity 5, and then flows out from the outlet 42, and finally discharged through the waste liquid port 9. A constant pressure pump is used to apply negative pressure at the end of the outlet pipe to make the buffer solution flow into the main channel of the reaction chamber. By using the negative pressure injection method to reduce the gas residue in the system, the injection is rapid.

[0093] 5) Use a computer to record the signal collected by the data acquisition device in real time, and use this signal to observe the progress of the reaction: when the resonant frequency is stable, add a certain concentration ([L]1) of ATP solution (dissolved in binding buffer) to the system, and record the frequency-time curve and frequency change △f1; after the frequency is stable, add dissociation solution to the system to regenerate the interface; after the frequency returns to the initial baseline level, use the same method to detect the addition of two other concentration systems ([L]2 and [L]3), and obtain the frequency-time curve and frequency change (△f2 and △f3) at these two concentrations, respectively, as shown in Figure 5. Figure 5 By sequentially adding the ligand solution to be tested, the frequency-time curve corresponding to the ligand solution to be tested is obtained. The determination method is simple and quick, and does not require complicated instrument operation. It only needs to inject the sample after the output frequency is stable. A micro-cantilever beam sensor is used to extract multiple sets of experimental data at one time, avoiding the systematic errors introduced by multiple experiments and multiple instruments.

[0094] 6) If Figure 6 As shown, read Figure 5 Substitute the frequency change value △f corresponding to different ligand concentrations [L] into the formula We get [L] / △f=2.85×10 -3 [L]+3.85ⅹ10 -9 , the binding equilibrium constant K can be obtained A =7.42 x 10 -3 M -1 .

[0095] 7) The obtained K A Substitute into formula K D =1 / K A , the dissociation equilibrium constant K is calculated D =1.3μM.

[0096] 8) The obtained K A Substitute the formula △G°=-RTlnK A , the calculated Gibbs free energy △G°=-33.5kJ / mol.

[0097] 9) The obtained K A Substitute into the formula The surface coverage θ at each ATP concentration [L] was calculated.

[0098] 10) In Figure 5 Read the slope b of the frequency-time curve after adding different ligand concentrations [L] to the system, combine the frequency change △f and the surface coverage θ, and substitute it into the formula In, get That is k a =660M -1 s -1 .

[0099] 11) The obtained k a Substitute into the formula k d =k a / K A , calculate the dissociation rate constant k d , k d =0.00089s -1 ,like Figure 7 shown.

[0100] 12) If Figure 8 As shown, at different T1 (299K) and T2 (293K), ATP solution with the same concentration [L] = 1 μM was added to the system, and the slopes b1 and b2 were read respectively and substituted into the formula Calculate and obtain the activation energy E a =38.0 kJ / mol.

[0101] The above embodiments of the present invention have the following beneficial effects:

[0102] 1. The present invention can directly and quickly obtain the required kinetic and thermodynamic parameters according to the frequency-time curve, and the method for determining the required data curve is simple and fast, without the need for complex instrument operation. Based on the micro-weighing detection mode of the micro-cantilever sensor, direct detection can be performed without labeling the ligand in the solution;

[0103] 2. The method proposed in the present invention can extract multiple kinetic and thermodynamic parameters at one time based on the data collected by a microcantilever sensor, avoiding the systematic errors introduced by multiple experiments and multiple instruments;

[0104] 3. The method proposed in the present invention does not require mass sensitivity calibration of the microcantilever sensor, which simplifies the experimental steps and improves the measurement efficiency;

[0105] 4. The microcantilever sensor used in the present invention has extremely high sensitivity, overcoming the limitation that other methods can only detect macromolecules, and is used to study the interaction mechanism between small molecules with low molecular weight and their nucleic acid aptamers at the interface.

[0106] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for determining kinetic and thermodynamic parameters at a solid-liquid interface, characterized in that: The following steps are involved: S100. Provide a frequency-time graph corresponding to each of a plurality of ligand solutions of different concentrations to be tested and a frequency-time graph of another ligand solution of a concentration to be tested at two different temperatures, wherein the plurality of ligand solutions of different concentrations to be tested are at least three ligand solutions of different concentrations to be tested; S200. Calculate the kinetic and thermodynamic parameters at the solid-liquid interface according to the frequency-time curves corresponding to the various ligand solutions of different concentrations to be tested, wherein the parameters include at least the binding equilibrium constant K A , dissociation equilibrium constant K D , Gibbs free energy △G°, surface coverage θ, binding rate constant k a and the dissociation rate constant k d ; S300. Calculate the reaction activation energy E according to the frequency-time curve of another ligand solution of the concentration to be tested at the two different temperatures. a ; The frequency-time curve diagram includes the following acquisition methods: The micro-cantilever chip is placed in the detection system and sealed; the micro-cantilever sensor adopts a micro-weighing detection mode; The injection channel of the injection valve is transferred into the buffer solution, and the constant pressure pump is turned on. The system becomes negative pressure, and the buffer solution passes through the injection port, fills the detection cavity, and then flows out from the outlet, and finally discharged through the waste liquid port; Record the signals collected by the data collector in real time; When the resonant frequency is stable, add ATP solution with a concentration of [L]1 to the system, and record the frequency-time curve and frequency change; after the frequency is stable, add dissociation solution to the system to regenerate the interface; After the frequency returned to the initial baseline level, the two concentration systems of [L]2 and [L]3 were tested using the same method to obtain the frequency-time curves and frequency changes at [L]2 and [L]3 concentrations, respectively.

2. A method for determining kinetic and thermodynamic parameters at a solid-liquid interface according to claim 1, characterized in that: The kinetic and thermodynamic parameters at the solid-liquid interface are calculated based on the frequency-time curves corresponding to the various ligand solutions with different concentrations to be tested, and the parameters at least include the binding equilibrium constant K A , dissociation equilibrium constant K D , Gibbs free energy △G°, surface coverage θ, binding rate constant k a and the dissociation rate constant k d include: S201. Reading frequency change values ​​△f corresponding to the various different concentrations of the ligand to be tested according to the frequency-time curves corresponding to the various different concentrations of the ligand to be tested; S202. Calculate the binding equilibrium constant K of the reaction according to the frequency change values ​​△f corresponding to the various concentrations of the ligand to be tested. A ; S203. According to the binding equilibrium constant K of the reaction A , calculate the dissociation equilibrium constant K of the reaction D ; S204. According to the binding equilibrium constant K of the reaction A , calculate the Gibbs free energy of the reaction △G°; S205. According to the binding equilibrium constant K of the reaction A , the surface coverage θ corresponding to various different concentrations of the ligand to be tested is calculated; S206. Calculate the binding rate constant k of the reaction according to the frequency-time curves and the corresponding surface coverage θ corresponding to the various ligand solutions of different concentrations to be tested. a ; S207. According to the binding rate constant k of the reaction a , the dissociation rate constant k is calculated d .

3. A method for determining kinetic and thermodynamic parameters at a solid-liquid interface according to claim 2, characterized in that: The binding equilibrium constant K of the reaction is calculated based on the frequency change values ​​Δf corresponding to the various concentrations of the ligand to be tested. A include: According to the frequency change values ​​△f corresponding to the various concentrations of the ligands to be tested, the formula Calculate the binding equilibrium constant K of the reaction A ; Where [L] is the concentration of the ligand to be tested, and A is a constant reflecting the maximum number of reactive sites on the interface.

4. A method for determining kinetic and thermodynamic parameters at a solid-liquid interface according to claim 2, characterized in that: The binding equilibrium constant K according to the reaction A , calculate the dissociation equilibrium constant K of the reaction D include: According to the binding equilibrium constant K of the reaction A , through the formula K D =1 / K A Calculate the dissociation equilibrium constant K of the reaction D .

5. A method for determining kinetic and thermodynamic parameters at a solid-liquid interface according to claim 2, characterized in that: The binding equilibrium constant K according to the reaction A , the calculated Gibbs free energy of the reaction △G° includes: According to the binding equilibrium constant K of the reaction A , through the formula △G°=-RTlnK A The Gibbs free energy of the reaction △G° was calculated; Where R is the ideal gas constant, R=8.314J / (mol·K), and T is the absolute temperature (K) at which the experiment is conducted.

6. A method for determining kinetic and thermodynamic parameters at a solid-liquid interface according to claim 2, characterized in that: The binding equilibrium constant K according to the reaction A , the surface coverage θ corresponding to various different concentrations of the ligand to be tested is calculated, including: According to the binding equilibrium constant K of the reaction A , through the formula The surface coverage θ corresponding to various different concentrations of the ligand to be tested was calculated.

7. A method for determining kinetic and thermodynamic parameters at a solid-liquid interface according to claim 2, characterized in that: The binding rate constant k of the reaction is calculated based on the frequency-time curves and the corresponding surface coverage θ corresponding to the various ligand solutions with different concentrations to be tested. a include: S2061. According to the frequency-time curves corresponding to the various ligand solutions with different concentrations to be tested, the slopes b of the frequency-time curves corresponding to the various ligand solutions with different concentrations to be tested are read; S2062. According to the frequency-time curve slope b corresponding to the plurality of different ligand concentrations to be tested, the frequency change value △f corresponding to the plurality of different ligand concentrations to be tested and the corresponding surface coverage θ, the formula The binding rate constant k of the reaction was calculated a .

8. A method for determining kinetic and thermodynamic parameters at a solid-liquid interface according to claim 2, characterized in that: The binding rate constant k of the reaction is a , the dissociation rate constant k is calculated d include: According to the binding rate constant k of the reaction a , through the formula k d =k a / K A The dissociation rate constant k was calculated. d .

9. A method for determining kinetic and thermodynamic parameters at a solid-liquid interface according to claim 1, characterized in that: The two different temperatures include a first preset temperature T1 and a second preset temperature T2, The reaction activation energy E is calculated based on the frequency-time curve of the ligand solution of another concentration to be tested at the two different temperatures. a include: S301. According to the frequency-time curves of another ligand solution of the measured concentration at the two different temperatures, obtain the slope b1 of the frequency-time curve at the first preset temperature T1 and the slope b2 of the frequency-time curve at the second preset temperature T2; S302. Calculate the activation energy E according to the first preset temperature T1, the second preset temperature T2, the slope b1 of the frequency-time curve at the first preset temperature T1, and the slope b2 of the frequency-time curve at the second preset temperature T2. a .

10. A method for determining kinetic and thermodynamic parameters at a solid-liquid interface according to claim 9, characterized in that: The activation energy E is calculated based on the first preset temperature T1, the second preset temperature T2, the slope b1 of the frequency-time curve at the first preset temperature T1, and the slope b2 of the frequency-time curve at the second preset temperature T2. a include: According to the first preset temperature T1, the second preset temperature T2, the slope b1 of the frequency-time curve at the first preset temperature T1, and the slope b2 of the frequency-time curve at the second preset temperature T2, the formula The activation energy E is calculated a ; Where R is the ideal gas constant, R=8.314J / (mol·K).

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